NCK RECRUITMENT TO EPH RECEPTOR, EPHB1 ELK, COUPLES LIGAND ACTIVATIONTO C-JUN KINASE/

Eph family receptor tyrosine kinases signal axonal guidance, neuronal bundling, and angiogenesis; yet the signaling systems that couple thes e receptors to targeting and cell-cell assembly responses are incomple tely defined. Functional links to regulators of cytoskeletal structure are anticipated based on receptor mediated cell-cell aggregation and migratory responses. We used two-hybrid interaction cloning to identif y EphB1-interactive proteins. Six independent cDNAs encoding the SH2 d omain of the adapter protein, Nck, were recovered in a screen of a mur ine embryonic library. We mapped the EphB1 subdomain that binds Nck an d its Drosophila homologue, DOCK, to the juxtamembrane region. Within this subdomain, Tyr(594) was required for Nck binding, In P19 embryona l carcinoma cells, activation of EphB1 (ELH) by its ligand, ephrin-B1/ Fc, recruited Nck to native receptor complexes and activated c-Jun kin ase (JNK/SAPK), Transient overexpression of mutant EphB1 receptors (Y5 94F) blocked Nck recruitment to EphB1, attenuated downstream JNK activ ation, and blocked cell attachment responses, These findings identify Nck as an important intermediary linking EphB1 signaling to JNK.